1. Field
Embodiments of the invention relate to the field of biomolecule 130 analysis. In particular, to systems, compositions and methods relating to rolling circle amplification and Raman detection of biomolecules 130.
2. Background
The sensitive and accurate detection, identification and/or quantification of low concentrations of biomolecules, such as proteins, peptides, oligonucleotides, nucleic acids, lipids, polysaccharides, hormones, neurotransmitters, metabolites, etc. has proven to be an elusive goal, with widespread potential uses in medical diagnostics, pathology, toxicology, epidemiology, biological warfare, environmental sampling, forensics and numerous other fields. Present detection methods typically involve detection of a fluorescently tagged capture molecule or ligand that can bind to the target biomolecule. Fluorescently tagged antibodies or probe oligonucleotides are typically used to bind to and detect target proteins or nucleic acids, respectively. Cross-reactivity and non-specific binding may complicate fluorescent detection of biomolecules in complex samples. Even where high specificity is obtained, the sensitivity of fluorescent detection is often insufficient to identify low concentrations of biomolecules. This is particularly true when the biomolecule to be detected is present at low concentrations in a complex mixture of other molecules, where interference, fluorescence quenching and high background fluorescence may all act to obscure or diminish the signal from the target biomolecule.
Other detection modalities have been attempted for biomolecule detection, such as Raman spectroscopy and/or surface plasmon resonance. When light passes through a tangible medium, a certain amount becomes diverted from its original direction, a phenomenon known as Raman scattering. Some of the scattered light also differs in frequency from the original excitatory light, due to the absorption of light and excitation of electrons to a higher energy state, followed by light emission at a different wavelength. The wavelengths of the Raman emission spectrum are characteristic of the chemical composition and structure of the light absorbing molecules in a sample, while the intensity of light scattering is dependent on the concentration of molecules in the sample.
The probability of Raman interaction occurring between an excitatory light beam and an individual molecule in a sample is very low, resulting in a low sensitivity and limited applicability of Raman analysis. It has been observed that molecules near roughened silver surfaces show enhanced Raman scattering of as much as six to seven orders of magnitude. This surface enhanced Raman spectroscopy (SERS) effect is related to the phenomenon of plasmon resonance, wherein metal nanoparticles exhibit a pronounced optical resonance in response to incident electromagnetic radiation, due to the collective coupling of conduction electrons in the metal. In essence, nanoparticles of gold, silver, copper and certain other metals can function as miniature “antenna” to enhance the localized effects of electromagnetic radiation. Molecules located in the vicinity of such particles exhibit a much greater sensitivity for Raman spectroscopic analysis.
Attempts have been made to exploit SERS for biomolecule detection and analysis, typically by coating metal nanoparticles or fabricating rough metal films on a substrate and then applying a sample to the metal-coated substrate. However, the number of metal particles that can be deposited on a planar substrate is limited, producing a relatively low enhancement factor for SERS and related Raman techniques utilizing such substrates. Thus, present methods of SERS detection do not exhibit sufficient sensitivity to detect low concentrations of biomolecules. A need exists for highly sensitive and specific methods of biomolecule detection.